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Abstract

The ability to utilize different selectable markers for tagging or mutating multiple genes in Schizosaccharomyces pombe is hampered by the historical use of only two selectable markers, ura4+ and kanMX6; the latter conferring resistance to the antibiotic G418 (geneticin). More markers have been described recently, but introducing these into yeast cells often requires strain construction from scratch. To overcome this problem we and other groups have created transformation cassettes with flanking homologies to ura4+ and kanMX6 which enable an efficient and time-saving way to exchange markers in existing mutated or tagged fission yeast strains.

Here, we present a protocol for single-step marker switching by lithium acetate transformation in fission yeast, Schizosaccharomyces pombe. In the following we describe how to swap the ura4+ marker to a kanMX6, natMX4, or hphMX4 marker, which provide resistance against the antibiotics G418, nourseothricin (clonNAT) or hygromycin B, respectively. We also detail how to exchange any of the MX markers for nutritional markers, such as arg3+, his3+, leu1+ and ura4+.

This single-step marker swap protocol for Schizosaccharomyces pombe allows for any tagged or mutated gene marked with an MX-type antibiotic marker to be swapped for a nutritional marker (cassettes containing the arg3+, his3+, leu1+, and ura4+ have been constructed) and to exchange genetic ura4+-markers for any MX-type antibiotic resistance marker (kanMX, natMX, and hphMX constructs have been tested for this study) (Lorenz et al., 2015a). Previously, this kind of approach was only feasible for MX-type antibiotic resistance markers (Sato et al., 2005; Hentges et al., 2005). Exchanging antibiotic resistance markers for each other already represented a basic set of useful genetic tools, the ura4+-to-MX as well as the arg3MX4, his3MX4, leu1MX4, and ura4MX4 marker swap cassettes expand this genetic toolbox for tagging and mutating genes in fission yeast (Lorenz et al., 2015a). The lithium acetate transformation protocol itself was described previously (Keeney and Boeke, 1994) and recently suggested modifications (http://listserver.ebi.ac.uk/pipermail/pombelist/2014/004012.html) were incorporated to provide a highly efficient procedure. Streamlining Schizosaccharomyces pombe strain construction in this way is time-saving and, therefore, will prove useful for fission yeast researchers.

Appropriate Schizosaccharomyces pombe strains: for a marker swap the strain must already carry a mutant or tagged gene marked with either an ura4+ or MX-type marker, such as kanMX, natMX, or hphMX (Bähler et al., 1998; Goldstein and McCusker, 1999). When introducing arg3MX4, his3MX4, leu1MX4, or ura4MX4 into a strain, this strain needs to be mutated for the respective gene at its original locus, e.g., arg3-D4 (Waddell and Jenkins, 1995), his3-D1 (Burke and Gould, 1994), leu1-32 (Keeney and Boeke, 1994), or ura4-D18 (Grimm et al., 1988).

Generation of marker swap cassettes for transformation
Marker swap cassettes to be used in the lithium acetate protocol below can be generated by two alternative means; by PCR amplification from or by restriction endonuclease digestion of the plasmids previously described in Lorenz (2015a and 2015b) (see below). Using appropriate modifications, this protocol can also be applied to constructs described elsewhere (Sato et al., 2005; Hentges et al., 2005; Gadaleta et al., 2013; Chen et al., 2015). After the PCR or restriction digest run 1/20 volume of each reaction on a 0.8% agarose gel in 1x TAE at 80 V for 45 min to verify that the reaction has worked properly (band sizes to be expected for each reaction are detailed below). The remainder of the PCR reaction or restriction is stopped by the addition of 2 μl of 0.5 M EDTA (pH 8.0). DNA concentration is measured on a NanoDrop 2000c UV/Vis-spectrophotometer, and an appropriate volume (maximum 20 μl) to result in 1-5 μg of cassette DNA is used for each transformation.

Grow 100 ml of yeast cells from the strain(s) to be transformed in YES broth in a shaking incubator at 30 °C to a density of ~1 x 107 cells/ml (count with haemocytometer to establish density) (see Notes 2 and 3).

Replica-plate onto a fresh selective plate and onto a plate selecting for the original marker, to ensure that the marker swap is correct (e.g., if the original strain was ura4+ and pALo120 was used to swap to kanMX6, the resulting strain must be resistant to G418 and unable to grow on media lacking uracil).

Data analysis

The critical step of the single-step marker swap procedure is to confirm that the markers have been truly exchanged in a transformant, i.e., that the new marker is correctly integrated at the target site, thereby removing the original marker. It is known that marker integration at its intended target site is not perfectly efficient in Schizosaccharomyces pombe, and a wide range of correct integration frequencies have been reported (Bähler et al., 1998; Sato et al., 2005). Correct integration is influenced by several parameters, including the chromatin status of the genomic site and length of sequence homologies flanking the marker cassettes. In the presented single-step marker swap cassettes flanking sequence homologies are between 200 and 400 bps, which is longer than the required minimum 80 bps (Bähler et al., 1998), but not long enough to enable 100% correct targeting in all cases. Swapping the ura4+ marker to any of the antibiotic MX markers occurred at frequencies between 22.9-100%. The correct integration efficiency of swapping an MX marker to a nutritional marker was tested at the meiotic gene hop1; this is more challenging due to the closed chromatin state at meiotic open reading frames (Bähler et al., 1998). Therefore, the frequency of exchanging a kanMX6-marker deleting hop1 with a nutritional marker was lower and varied between 15.3-36.6% (Lorenz, 2015a).

Notes

When pipetting a PCR reaction or restriction digest, always start with the MilliQ water, add the buffer, then all other components, and finally the enzyme(s). Working from these protocols, it is important to calculate the amount of water required before one sets up the reaction.

For the transformation protocol use yeast cells streaked onto the appropriate plate not longer than 7 days beforehand. Always check the genotype of yeast strains carefully before usage!

It is crucial to grow the fission yeast cells to late logarithmic phase (~1 x 107 cells/ml) to achieve the most efficient transformation frequency. Growing cells to higher density will result in drastically reduced numbers of transformants, because cells enter stationary phase and develop a thicker cell wall (i.e., do not grow cells to higher than ~1 x 107 cells/ml and dilute).

40% PEG (see Recipes) needs to be prepared freshly on the day when the transformation is performed.

The duration and temperature
of the heat shock (step B11) is critical!

We acknowledge funding from the Biotechnology and Biological Sciences Research Council UK (BBSRC, Doctoral Training Grant BB/FO16964/1) and the College of Life Science and Medicine, University of Aberdeen, UK.